SUMMARY (PROJECT 1) Anti-CD19 chimeric antigen receptor redirected T cells (CART-19) have been dramatically successful for some patients with B-cell malignancies. We and others have shown complete response (CR) rates of over 90% in patients with relapsed/refractory (r/r) ALL. Many of these remissions are sustained, but the major limitation is relapse in 20-50% of patients and two-thirds of these relapses involve CD19-negative ALL. In patients with r/r CLL treated with anti-CD19 CAR T cells, complete response rates are lower at 20-25%, but relapse after remission is very unusual. In addition, there are also many patients with B cell malignancies who cannot benefit from CAR T cells because of the inability to collect or manufacture T cells, especially from intensively treated patients with low T cell counts and from infants. In addition, some patients will rapidly progress before manufacturing is complete and cannot tolerate or survive the necessary treatment delay. This project will use a comprehensive strategy to target the major limitations in CAR T cell therapy, increasing the feasibility of this already transformational approach by looking both at T cell-intrinsic and extrinsic mechanisms of resistance as well as ALL cell-intrinsic mechanisms that lead to relapse. We will take advantage of already established successful collaborations with core laboratories with expertise in gene editing, as well as state of the art correlative science to explore a number of innovative aims. We have designed studies to understand the biology and mechanisms underlying both CD19 negative and CD19 positive relapse in ALL. In CLL, where response rates are a more significant barrier than relapse, we will explore the role that checkpoint molecules, the tumor microenvironment, and the leukemia cell itself plays in inducing resistance to CTL019. To do this, we will capitalize on our unique bank of specimens derived from over 200 patients treated at Children's Hospital and U Penn with CTL019. Finally, we have designed high impact clinical trials 1) testing methods to limit CD19 negative relapse in ALL (by targeting CD19 and CD22 concurrently) and 2) enhancing responses in B cell malignancies by testing gene-edited, CAR modified allogeneic T cells (?PACE? CARs). PACE CARs are pre-manufactured, ?universal donor? CAR+ cells that are gene-edited with CRISPR technology to eliminate T cell receptors and HLA class I molecules. These cells could be used across HLA barriers and should not cause GVHD or be rapidly rejected. Therefore, they can solve unmet medical needs in autologous CAR therapy where i) there is a high degree of clinical urgency (cells are immediately available); ii) there has been a failure to collect adequate T cells or manufacture autologous CAR cells; and iii) where autologous CTL019 has failed to induce B cell aplasia and/or clinical response. This research will be highly significant because it will lead to detailed understanding of mechanisms of resistance to CTL019, provide information that can be rapidly translated for clinical testing, and perform highly innovative clinical trials attempting to enhance outcomes for patients who currently have no effective treatment options.